The discovery and development of a vaccine candidate that elicits a broad-spectrum protection against diverse influenza subtypes, also termed a universal vaccine, is needed to prevent or limit the spread of influenza viruses with pandemic potential in pigs and humans. Some progress has recently been made in generating a universal vaccine, such as M2e and virus-like-particle vaccines. Perhaps the most significant progress is the discovery of CR6261, F10, and 12D1 antibodies apparently targeting viral fusion machinery largely constituted by HA2, rather than the receptor-binding site in HA1. CR6261 and F10 bind to conformational epitopes within the conserved A helix of HA2 likely presented in the context of the adjoining region, while 12D1 binds to a linear eptiope within the long CD helix of HA2. However, such antibodies are infrequently induced either by immunization with influenza vaccines or through natural infection, suggesting that their epitope(s) are temporary and short-lived, or are spatially restricted in exposure. The goal of this R15 project is to identify immunogens that elicit a broadly neutralizing antibody response targeting both the A helix and the CD helix of HA2. This will be achieved by modifying the HA2 protein to allow exposure of the A helix as well as the CD helix with its adjoining region in a native context that will preserve a unique set of broadly neutralizing epitopes represented by CR6261/F10/12D1. Specifically, we will generate the fusion intermediates that present these conserved targets to the immune system as they exist after low pH triggering, but prior to fusogenic structure formation. We plan to utilize the virus-like-particle (VLP) platform as a technical basis to produce our innovative fusion-active vaccine. Another goal in this application is to evaluate the efficacy of M2e vaccine in a pig model and determine whether it induces protective immunity, or an adverse immune response as was demonstrated in a previous study. This R15 project will be accomplished by: Aim 1, To prepare, characterize, and purify H1N1 fusion-active and M2e immunogens, and Aim 2, To evaluate selected immunogens and vaccination strategies in a pig model for their ability to elicit protective immune responses against different subtypes of influenza A virus. This project will be approached by an interdisciplinary team that consists of David Francis (Co-PI, Pig model), Radhey Kaushik (Co-PI, swine immunology), Feng Li (Investigator, influenza virology) and two swine influenza consultants (Weijun Ma and Mahesh Khatri). PUBLIC HEALTH RELEVANCE: Influenza remains a significant cause of morbidity and mortality worldwide with the potential to cause pandemics. The vaccine is by far the most effective means of preventing influenza infection;however, rapid genetic drift and reassortment associated with viral surface glycoproteins often render vaccine less effective particularly against virus strains not closely related to vaccine strains, which has been highlighted in 2009 swine influenza H1N1 pandemic. Thus, the development of new vaccine strategies eliciting potent and broadly cross-protective antibodies is urgently needed, and has been viewed as a global public health priority. The goal of the proposed research is to identify immunogens that will elicit a broadly neutralizing antibody response in a pig model against different subtypes of influenza viruses. If successful, the immunogens studied in this project will provide critical insight to develop a universal influenza vaccine that can used to prevent or limit the spread of influenza viruses with pandemic potential in both pigs and humans.